Interleukin‐33 increases type 2 innate lymphoid cell count and their activation in eosinophilic asthma

Abstract Background Interleukin‐33 (IL‐33) exacerbates asthma probably through type 2 innate lymphoid cells (ILC2s). Nevertheless, the association between eosinophilic asthma (EA) and ILC2s remains obscure, and the mechanisms by which IL‐33 affects ILC2s are yet to be clarified. Methods ILC2s were evaluated in peripheral blood mononuclear cells, induced sputum, and bronchoalveolar lavage fluid obtained from patients with EA. Confocal microscopy was performed to locate ILC2s in lung tissue and the mRNA expression of ILC2‐related genes was also evaluated in the EA model. The proliferation of ILC2s isolated from humans and mice was assessed following IL‐33 or anti‐IL‐33 stimulation. Results The counts, activation, and mRNA expression of relevant genes in ILC2s were higher in PBMCs and airways of patients with EA. In addition, ILC2 cell counts correlated with Asthma control test, blood eosinophil count, Fractional exhaled nitric oxide level, and predicted eosinophilic airway inflammation. IL‐33 induced stronger proliferation of ILC2s and increased their density around blood vessels in the lungs of mice with EA. Moreover, IL‐33 treatment increased the counts and activation of ILC2s and lung inflammatory scores, whereas anti‐IL‐33 antibody significantly reversed these effects in EA mice. Finally, IL‐33 enhanced PI3K and AKT protein expression in ILC2s, whereas inhibition of the PI3K/AKT pathway decreased IL‐5 and IL‐13 production by ILC2s in EA. Conclusions ILC2s, especially activated ILC2s, might be critical markers of EA. IL‐33 can induce and activate ILC2s in the lungs via the PI3K/AKT pathway in EA. Thus, using anti‐IL‐33 antibody could be a part of an effective treatment strategy for EA.


| INTRODUCTION
Asthma, a prevalent chronic airway illness, is classified into the eosinophilic asthma (EA), neutrophilic asthma, mixed asthma, and paucigranulocytic asthma based on the counts of eosinophils and neutrophils in the sputum. Patients with EA exhibit worse symptoms than those with non-EA (non-eosinophilic asthma (NEA)), regardless of asthma severity. 1 Recent studies have confirmed the crucial involvement of innate lymphoid cells (ILC2s), a rare group of lymphocytes, in innate immune responses to allergic diseases. 2 Innate lymphoid cells lack antigen recognition receptors and produce IL-13 and IL-5, both of which are involved in EA pathogenesis. 3 Interleukin-33, which is produced by bronchial epithelial cells, is crucial for the activation of resident lung Th2 cells as well as ILC2s to produce IL-5, resulting in the development of chitin-induced airway eosinophilia. 4 In addition, IL-33 and IL1RL1 (ST2) variants are reported to be significantly associated with asthma. [5][6][7] Studies have established the correlation of a higher number of ILC2s in the blood, sputum, or bronchoalveolar lavage fluid (BALF) in allergic asthma, and the increased number of ILC2s has been related to asthma exacerbation. [8][9][10] In the lungs, ILC2s, along with dendritic cells and regulatory T cells, localize to the lung bronchi and larger vessels. 11 However, the localization of ILC2s in the lungs has been mostly studied in transgenic mice, which limits the interpretation of the results and their application to humans. Moreover, only a limited number of studies have documented the association between EA and ILC2s.
In patients with allergic asthma, increased levels of ILC2s are linked to increased eosinophils. 12 IL-33 is crucial for the activation of ILC2s. 13 Notably, the p38 mitogen-activated protein kinase (MAPK), NF-κB, PI3K/Akt, and Wnt/β-catenin pathways regulate the transcription of IL-33 in murine and human endothelial cells 14 and activation of the PI3K/AKT signaling pathway induces biological processes in eosinophils. 15 Therefore, additional studies are required to address the mechanisms of IL-33 in ILC2s in EA. This study aimed to assess the level and function of ILC2s in patients with EA as well as their correlation with IL-33.

| Study design and subjects
This study was approved by the Ethics Committee of the Fifth Affiliated Hospital of Sun Yat-sen University (approval no. K35-1).
Informed consent was obtained from 255 patients with asthma and 54 healthy controls (HCs). After applying inclusion and exclusion criteria, 203 patients with asthma and 40 healthy individuals were enrolled in the study. Supplementary Figure S1 shows a detailed description of the research design.

| Induced sputum and classification of inflammatory cells
Sputum specimens were obtained as previously reported. 16 Individuals with ≥3% eosinophils in sputum were considered to have EA. A detailed description is provided in the supplementary material. and IL-13+ILC2s, respectively. Samples were subjected to flow cytometry (BD laser II) and analyzed using the Kaluza software (Beckman, BD, USA).

| Pathological staining and inflammatory cell count
Mice lung tissue embedded in paraffin were sliced, dewaxed, dehydrated in a graded series of ethanol, and stained with hematoxylin-eosin staining. Bronchus-bronchial lesions were assessed and scored under a light microscope according to a previous method. 17 A detailed description is provided in the supplementary material.  Enzyme-linked immunosorbent assay   IL-5, IL-33 and IgE in the plasma of the subject and IL-5, IL-13 and   OVA-IgE in the culture supernatant of ILC2s isolated from lung tissue   in mice were determined using ELISA kits (MEIMIAN, Wuhan, China) according to the manufacturer's protocol.

| Location of ILC2s in lung tissue using confocal microscopy
Briefly, 4-μm-thick murine lung tissue slices were dewaxed and rehydrated in a graded series of ethanol. Next, their endogenous peroxidase activity was blocked and the sections were subjected to epitope retrieval, following which, they were incubated with primary antibodies against Anti-mouse CD3e-biotin (dilution 1:100 eBio500A2, eBioscience,Cal, USA), Anti-rat GATA3 (dilution 1:50,

| Evaluation of ILC2-related gene expression using RT-qPCR
Total RNA was extracted from human peripheral blood or mouse lung tissue using TRIzol LS (Thermo Fisher Scientific, Waltham, MA, USA). A detailed description and primer sequences are provided in the supplementary material.

| Sorting of ILC2s from human PBMCs and murine lung tissues and evaluation of proliferation of ILC2 in vitro
PBMCs were isolated from individuals in the HC and EA groups. In addition, lungs were resected from mice in the NC and EA groups.
Innate lymphoid cells were sorted using magnetic beads (STEMCELL, BC, Canada); this process involved the isolation of CRTH2+ cells using column-free immunomagnetic-positive sorting. Finally, an ILC2 isolation cocktail was added, and the enriched cell suspension was transferred to a fresh tube using a magnet; non-ILC2s were discarded. The process of ILC2 sorting in the lungs of mice is described in the supplementary material. Sorted ILC2s were cultured and relative cell viability was calculated as reported previously. 18 A detailed description is provided in the supplementary material.

| Western blot analysis
Innate lymphoid cells were sorted from the OVA+IL-33 group and exposed to the AKT inhibitor AZD5363 (10 μmol/L; Selleck, Shanghai, China) for 24 h. A detailed description is provided in the supplementary material. Count data were analyzed using X 2 test. Spearman's rank correlation was used to analyze the correlations between ILC2s and other factors. Mann-Whitney test was used to analyze the lung function index.

| Statistical analyses
Statistical significance was set at p < 0.05.

| Number and associated gene expression of ILC2s increased in EA
Innate lymphoid cells have been reported to express CRTH2 and CD127. 19 In our study, ILC2s isolated from PBMCs and induced sputum (IS) of patients with asthma were sorted into the CD45 + Lin − CD127 + CRTH2 + population ( Figure 1A-D). In particular, patients with asthma had higher numbers of ILC2s in PBMCs and sputum than the HC group (p < 0.05). Moreover, patients with EA had higher counts of ILC2s than those with NEA (p < 0.05, Figure 1B,D) and higher levels of IL-5 and IL-33 than those with NEA (p < 0.05, Fig. E and F). Furthermore, an increased proportion of IL-5 + ILC2s and IL-13 + ILC2s was observed in PBMCs of patients with asthma, particularly in the EA group ( Figure S2). We also detected that the expression of GATA3 and RORa was remarkably higher in the EA group compared with that in other groups ( Figure S3A, B). However, CD127 expression did not differ between the groups ( Figure S3C). The CRTH2 and ICOS mRNA levels were the highest in the EA group (PBMCs, Figure S3D, E), while KLRG1 expression was increased in the EA group ( Figure S3F). Finally, the IL-33 mRNA level in the blood of EA patients and lung tissue of EA mice was remarkably higher than those in other groups ( Figure S3G, Figure S7F). SUN ET AL.
-3 of 13 F I G U R E 1 The innate lymphoid cell (ILC2) numbers were elevated in EA (A) ILC2s were gated as the CD45 + Lin − CD127 + CD294 + population in PBMCs obtained from HC and patients with asthma. (B) The numbers of ILC2s were significantly higher in PBMCs obtained from the EA group (n = 30) than those from the HC and NEA groups. (C) ILC2s were gated as the CD45 + Lin − CD127 + CD294 + population in induced sputum (IS) obtained from the HC group and patients with asthma. (D) The number of ILC2s was significantly higher in the IS of the EA group. (E-F) The levels of IL-5 and IL-33 were significantly higher in the serum of the EA group. HC, healthy control; EA, eosinophilic asthma; NEA, non-eosinophilic asthma. ▽Comparison with HC group, ▽p < 0.05, ▽▽p < 0.01; ▽▽▽p < 0.001; *Comparison with EA group, *p < 0.05, **p < 0.01. The Kruskal-Wallis test was used to analyze statistical significance between groups.
3.2 | Number of ILC2s correlated with ACT, blood eosinophil count, and FeNO levels, and predicted eosinophilic airway inflammation Table 1 shows the baseline characteristics of the individuals enrolled in our study. The EA group had the highest counts of blood eosinophils and basophils (BASs). In addition, the EA group was characterized by a higher incidence of allergy history and omalizumab usage, increased Fractional exhaled nitric oxide (FeNO) level, and specific and total IgE, but lower Asthma control test (ACT) scores than those in other groups ( Table 1). The EA group presented the lowest FEV1% pred and the highest Rc, Rp, Z5, R5, R20, and Fres among the four groups (Table S1 and Figure S4). Furthermore, the percentage of (r s = 0.71, p = 0.005), but negatively correlated with ACT (r s = 0.382, p = 0.037). We did not detect any correlation between IgE, FEV1%, and FEV1/forced vital capacity (%) (p > 0.05) ( Figure 2). Furthermore, using receiver operating characteristic analysis, we identified biomarkers of respiratory eosinophilic inflammation (sputum eosinophil count ≥3%). We found that ILC2s number was a sensitive EA diagnostic biomarker. Using a threshold value of ≥0.065% for ILC2s and ≥0.465 � 10 9 for eosinophils, we could distinguish between eosinophilic and non-eosinophilic inflammation with high sensitivity and specificity (89% and 50%, respectively, Table S2). Moreover, the area under the curve of blood eosinophils and FeNO for differentiating between eosinophilic and noneosinophilic inflammation was 0.708 and 0.741, respectively (p = 0.05, p = 0.028; Figure S5) However, IgE level and ACT score could not distinguish between eosinophilic and non-eosinophilic inflammation (Table S2). These factors were selected based on the Spearman rank test.

| IL-33 induced proliferation and pulmonary accumulation of ILC2s in EA
We isolated ILC2s from the peripheral blood of patients in the EA and HC groups at 91% purity ( Figure 3A,B). In addition, we obtained ILC2s from the lung tissue of EA and NC mice with a purity of up to 94% ( Figure 3D,E). We found that ILC2s from the control and EA groups proliferated rapidly when cocultured with rIL-33 in vitro. However, we did not detect any difference in the in vitro proliferation of ILC2s obtained from the control and EA groups. Interestingly, ILC2s obtained from the EA group showed stronger in vitro proliferation after treatment with rIL-33 compared with those from other groups ( Figure 3C, D). We also examined the distribution and proliferation of ILC2s in vivo. We labeled and observed ILC2s (CD3-GATA3+ICOS+) gathered around lung blood vessels using confocal microscopy and found an increased accumulation of ILC2s around the blood vessels of murine lungs in the EA group following rIL-33 stimulation ( Figure 4A,B).

| IL-33 enhanced the activation of IL-5+ILC2s and IL-13+ILC2s, the associated gene expression of ILC2s, and aggravated lung allergic inflammation in mice with EA
We observed that ILC2s in PBMCs and BALF of asthmatic mice gated as the CD45 + Lin − CD127 + ST2 + population ( Figure 5A,C). Moreover, Histopathological examination of the lungs revealed that the OVA +IL-33 group had the highest inflammation scores among the four groups of EA mice ( Figure 9A, B, p < 0.05).

| PI3K/AKT signaling pathway was involved in IL33/ILC2s
We isolated ILC2s from lung tissues to determine the effects of IL-33 on the PI3K/AKT signaling pathway. We found that IL-33 increased the ratio of p-PI3K/t-PI3K and p-AKT/t-AKT, whereas treatment with anti-IL-33 antibody decreased the ratio of p-PI3K/p-AKT in ILC2s obtained from EA mice ( Figure 6A). In addition, the administration of the AKT inhibitor AZD5363 reduced the expression of p-PI3K and p-AKT. Conversely, treatment with the AKT agonist SC79 increased the expression of p-PI3K and p-AKT ( Figure 6B). Furthermore, the AKT inhibitor AZD5363 reduced the levels of IL-5 and IL-13; in contrast, the AKT agonist SC79 significantly reversed the elevated levels of IL-5 and IL-13 ( Figure 6C-D).

| DISCUSSION
This study suggests that the number of ILC2s, especially those of from T-and B-cells by facilitating the proliferation and aggregation of pulmonary and intestinal ILC2s. 24 Our study revealed that ICOS mRNA expression was increased in EA, which enhanced the proliferation and accumulation of mature lung ILC2s. IL-33 also activates ILC2s in type 2 immunity. 25 Our study indicated that the increased mRNA expression of IL-33 is due to the increased activation of ILC2s in EA.
In our study, the eosinophil count positively correlated with ILC2 number, but negatively correlated with the ACT. We then explored the diagnostic potential of ILC2s in eosinophilic airway inflammation.
The number of eosinophils in IS contributes significantly to the diagnosis of eosinophilic airway inflammation. Nonetheless, patients with asthma tend to produce less sputum, making this procedure less The production of IL-5 and IL-13 in isolated ILC2s following treatment with an AKT inhibitor (AZD5363) or AKT agonist (SC79). NC, negative control; p-PI3K, phosphorylated PI3K; t-PI3K, total PI3K; p-AKT, phosphorylated AKT; t-AKT, total AKT; AZD5363, AKT inhibitor; SC79, AKT agonist. ▽Comparison with NC group, ▽p < 0.05; *Comparison between different stimulated groups, *p < 0.05, **p < 0.01,***p < 0.001. ANOVA test was used to analyze statistical significance between groups. ANOVA, analysis of variance. The mechanism underlying the contribution of IL-33 and ILC2s to asthma exacerbation has not yet been established in patients with EA. The p38 MAPK, NF-κB, PI3K/Akt, and Wnt/catenin pathways reportedly regulate IL-33 transcription in endothelial cells in mice and humans. 14 In patients with eosinophilic nasal polyps, IL-33 induces the production of IL-4 and IL-5 via the PI3K/AKT pathway. 33 Accordingly, PI3Kδ reduced the expression of IL-33 and number of ILC2s, thereby inhibiting the allergic inflammatory response. 34 Leptin, a small proline protein, and LAIR-1 regulate ILC2s by targeting the PI3K-AKT pathway, hence affecting the severity of asthma. [35][36][37] Therefore, we hypothesized that IL-33 and ILC2s play a role in asthma via the PI3K/AKT signaling pathway. In our study, IL-33 upregulated the expression of PI3K and AKT proteins in ILC2s through ST2, increasing the levels of IL-5 and IL-13 (Th2 cytokines) and aggravating lung inflammation in patients with EA. Therefore, we propose that the PI3K/AKT pathway plays a mediating role in IL33/ ILC2 and affects the subsequent inflammatory response.
Our study had certain limitations. We acknowledge that singlecenter enrollment can lead to sample bias. This was mainly an observational study, and as such, our results on the mechanism of